The physical environment of a living cell acutely determines its ability to proliferate, metabolize, differentiate and remodel. On the one hand, living cells specify lineage and express different phenotypic and physical states with extreme responsiveness to stiffness of their underlying matrix, while on the other, cell stretch, especially as occurs in cells resident in heart, lung, muscle, and gut, is a potent biological stimulus and regulator of tissue and cell size, structure and composition. In general, however, it has not been possible to reproduce both of these aspects of a cell's in-vivo microenvironment, viz. underlying substrate stiffness and cell stretch, within an in-vitro culture.
Techniques have been previously developed in order to reproduce cell stretch within in-vitro cultures. These methods may be broadly classified into the following categories: stretching the adherent cell's underlying substrate, applying hydrostatic pressure within the cell culture chamber, prescribing shear stresses via fluid flows over the adherent cells, applying localized loads using magnetic microbeads, microneedles, AFM cantilevers, micropipettes and optical tweezers, and micropipette and microplate manipulations of cell volume. Some of these techniques have been commercialized and used in biological research (e.g. Flexcell International, STREX from B-Bridge International).
The prior art systems, however, have generally ignored the role of underlying substrate stiffness, by culturing cells on substrates whose stiffness (˜109 Pa) is several orders of magnitude greater than that of the adherent cell. In general, in addition to mechano-sensitivity to external loads, diverse cell types also sense and respond to stiffness of their extra-cellular matrix by modulating their adhesions, shape, contractility, cytoskeletal structure and overall cell state. For example, when cultured on soft matrices (100 to 1000 Pa) that mimic stiffness of brain tissue, stem cells expressed a neurogenic phenotype. When cultured on intermediate stiffness substrates (8000-17000 Pa) resembling the stiffness of muscle, the stem cells expressed a myogenic phenotype. Also, when cultured on even stiffer substrates (25000-40000 Pa) that approximates the stiffness of collagenous bone, stem cells commit to an osteogenic phenotype. Some studies have reported considerable implications of local matrix stiffness on cell differentiation, proliferation, spreading and migration, mechanotransduction, osteogenesis and several disease processes.
Accordingly, the inventions described herein overcomes methodological limitations in the described prior art techniques that either culture cells on soft matrices that are static and passive, or on exceedingly stiff, dynamic substrates.